title: Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: implications for angiotensin II-induced vascular dysfunction.
abstract: Glutathionylation of the Na(+)-K(+) pump's β1-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na(+)-K(+) pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na(+)-K(+) pump activity is an important determinant of vascular tone through effects on [Ca(2+)]i, we have examined the role of oxidative regulation of the Na(+)-K(+) pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β1-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na(+)-K(+)ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K(+)-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na(+)-K(+) ATPase and decrease in K(+)-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β1-subunit. Knock-out of FXYD1 dramatically decreased K(+)-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8 mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β1-subunit glutathionylation, and enhanced K(+)-induced vasorelaxation. Ang II inhibits the Na(+)-K(+) pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump's β1-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na(+)-K(+) pump and may have an important protective role in the vasculature under conditions of oxidative stress.
authors.affiliation: North Shore Heart Research Group, Kolling Institute of Medical Research, University of Sydney, Australia.
authors.forename: Chia-Chi
authors.initials: CC
authors.lastname: Liu
authors.affiliation: North Shore Heart Research Group, Kolling Institute of Medical Research, University of Sydney, Australia; Department of Cardiology, Royal North Shore Hospital, Sydney, Australia.
authors.forename: Keyvan
authors.initials: K
authors.lastname: Karimi Galougahi
authors.affiliation: Vascular Biology Section, Department of Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Weisbrod
authors.affiliation: North Shore Heart Research Group, Kolling Institute of Medical Research, University of Sydney, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hansen
authors.affiliation: North Shore Heart Research Group, Kolling Institute of Medical Research, University of Sydney, Australia.
authors.forename: Ramtin
authors.initials: R
authors.lastname: Ravaie
authors.affiliation: North Shore Heart Research Group, Kolling Institute of Medical Research, University of Sydney, Australia.
authors.forename: Andrea
authors.initials: A
authors.lastname: Nunez
authors.affiliation: Laboratory Membrane Biology, Massachusetts General Hospital, Boston, MA, USA.
authors.forename: Yi B
authors.initials: YB
authors.lastname: Liu
authors.affiliation: North Shore Heart Research Group, Kolling Institute of Medical Research, University of Sydney, Australia.
authors.forename: Natasha
authors.initials: N
authors.lastname: Fry
authors.affiliation: North Shore Heart Research Group, Kolling Institute of Medical Research, University of Sydney, Australia.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Garcia
authors.affiliation: North Shore Heart Research Group, Kolling Institute of Medical Research, University of Sydney, Australia.
authors.forename: Elisha J
authors.initials: EJ
authors.lastname: Hamilton
authors.affiliation: Laboratory Membrane Biology, Massachusetts General Hospital, Boston, MA, USA.
authors.forename: Kathleen J
authors.initials: KJ
authors.lastname: Sweadner
authors.affiliation: Vascular Biology Section, Department of Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Cohen
authors.affiliation: North Shore Heart Research Group, Kolling Institute of Medical Research, University of Sydney, Australia; Department of Cardiology, Royal North Shore Hospital, Sydney, Australia. Electronic address: gemma.figtree@sydney.edu.au.
authors.forename: Gemma A
authors.initials: GA
authors.lastname: Figtree
mesh_terms: D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001158:Arteries; D005455:Fluorescent Antibody Technique; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D009129:Muscle Tonus; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D021122:Protein Subunits / Q000378:metabolism; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D000804:Angiotensin II; D019255:NADPH Oxidases; D000254:Sodium-Potassium-Exchanging ATPase; D005978:Glutathione
keywords: 4′,6-diamidino-2-phenylindole; ACE; Ang II; Angiotensin II; BCA; BioGEE; DAPI; DHE; DMSO; FITC; GSH; GSNO; Glutathionylation; KHB; Krebs-Henseleit buffer; NADPH oxidase; NO; NOX; Na(+)-K(+) pump; Oxidative signaling; PBS; PEG-SOD; Pi; RAS; S-nitrosoglutathione; VSMCs; Vascular tone regulation; [Ca(2+)](i); [Na(+)](i); angiotensin II; angiotensin converting enzyme; bicinchoninic acid; biotinylated glutathione ethyl ester; cGMP; cyclic guanidine monophosphate.; dihydroethidium; dimethyl sulfoxide; fluorescein isothiocyanate; glutathione; glutathionylated β(1)-subunit of Na(+)-K(+) pump; inorganic phosphate; intracellular calcium concentration; intracellular sodium concentration; nicotinamide adenine dinucleotide phosphate-oxidase; nitric oxide; pegylated superoxide dismutase; phosphate buffer saline; renin-angiotensin system; sGC; soluble guanylyl cyclase; vascular smooth muscle cells; β1-GSS